Nitroglycerin is widely used in the treatment of cardiovascular disease. A major limitation to the clinical use of nitroglycerin is nitrate tolerance, i.e. a rapid decline in vasodilation that occurs with continuous use. Our work has uncovered a novel, and potentially important, mechanism that serves to limit the degree of nitrate tolerance in arteries. Namely, that coincident with tolerance to the nitrates, blood vessels adapt by developing a compensatory relaxation pathway. This occurs through the increased expression of functional potassium channels that participate in nitrate-induced relaxations in tolerant, but not control blood vessels. This novel hypothesis is supported by functional and molecular biological preliminary data obtained with our animal model, in which rats are treated in vivo with nitroglycerin for several days. This leads to profound tolerance to nitroglycerin-induced relaxation of arteries studied ex-vivo, and provides an ideal model with which to further pursue mechanistic studies. The primary objective of this application is to critically characterize this compensatory mechanism by focusing on three major hypotheses. Firstly, we hypothesize that prolonged in vivo exposure to nitroglycerin increases BKCa channel gene and protein expression in vascular smooth muscle cells. Secondly, we hypothesize that the increase in BKCa channel expression results in an increase in the net nitrate-induced outward potassium current in tolerant smooth muscle cells. Thirdly, we hypothesize that increased expression and/or activity of functional BKCa channels results in an increased role for BKCa channels in smooth muscle relaxation of nitrate tolerant arteries. An important corollary hypothesis is that in tolerant vessels, this compensatory response will lead to a paradoxical increase in the efficacy of endogenous vasodilators or drugs that act primarily through BKCa channel activation (e.g. certain eicosanoids). The following Specific Aims are designed to investigate our hypotheses: In Aim #1, BKCa channel protein and mRNA expression will be determined in tolerant and nontolerant arteries. In Aim #2, patch-clamp recording will be used to quantify BKCa channel activity in tolerant and nontolerant arteries. In Aim #3, organ bath experiments will be used to evaluate the functional consequences of increased BKCa channel expression/activity in smooth muscle relaxation of nitrate tolerant arteries. The proposed studies will break new ground regarding our understanding of the vascular consequences of prolonged treatment with nitroglycerin, including the potential impact on normal homeostatic mechanisms that control blood vessel diameter (i.e. EDHFs and other endogenous BKCa channel activators). The knowledge gained from these studies may also provide insight into novel approaches for increasing BKCa channel activity in specific vascular beds and/or in certain conditions where BKCa channel expression is decreased (e.g. aging). PUBLIC HEALTH RELEVANCE: A major limitation to nitroglycerin therapy is the development of tolerance;i.e. rapid loss of efficacy with continuous use. By examining the role of potassium channels in nitroglycerin-induced vasodilation in normal vs. tolerant arteries, this project seeks to increase our understanding of how blood vessels adapt to prolonged exposure to nitroglycerin. The knowledge gained from these studies may suggest new strategies for increasing the number of potassium channels in specific blood vessels, treating conditions in which potassium channels are known to be diminished (e.g. aging), and developing novel drugs that may be particularly effective in nitrate tolerant patients.